FHL2 Is Essential for Spleen T Cell-Dependent B Cell Activation and Antibody Response.

Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule regulating various cellular processes, including signal transduction, transcription, and cell survival. Although involved in inflammation and immune responses, its role in the germinal center reaction and B cell maturation remains unknown. We found that FHL2-/- mouse spleens displayed enlarged follicles with more B cells. When a T cell-dependent immune response was elicited using SRBC, FHL2-/- germinal center area was enhanced 2-fold compared with wild type (WT), concomitant with expanded dark zones. Nevertheless, the SRBC-induced rise in spleen IgG1 expression, and plasma IgG1 levels observed in WT were absent in FHL2-/- mice, and circulating plasma cells were also reduced in FHL2-/- This could be explained by deficient upregulation of spleen activation-induced cytidine deaminase mRNA. Interestingly, FHL2-/- B cells successfully underwent class-switch recombination in vitro, and both activation-induced cytidine deaminase induction and IgG1 response to SRBC were equivalent in B cell-deficient µMT mice transplanted with WT or FHL2-/- bone marrow, suggesting that the defects observed in FHL2-/- mice were not B cell intrinsic. However, spleen lysates from FHL2-/- mice revealed a disturbed spleen microenvironment, with reduced CXCL12 and CXCL13 levels compared with WT. Our data suggest that spleen FHL2 expression is essential for a normal germinal center reaction and proper induction of class-switch recombination in response to a T cell-dependent Ag, leading to the emergence of Ab producing plasma cells. This could be due to the regulation of spleen cytokine production by FHL2.

LIM-domain transcription complexes interact with ring-finger ubiquitin ligases and thereby impact islet ß-cell function.

Diabetes is characterized by a loss of ß-cell mass, and a greater understanding of the transcriptional mechanisms governing ß-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA-binding protein 3 (SSBP3) regulates the genes necessary for ß-cell function, but few proteins are known to interact with this complex in ß-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse ß-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for ß-cell function. We observed that Rnf20 and Rnf40 depletion reduces ß-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate ß-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

miR-340-FHL2 axis inhibits cell growth and metastasis in ovarian cancer.

Although increasing evidence indicated that deregulation of microRNAs (miRNAs) contributed to tumor initiation and progression, but little is known about the biological role of miR-340 in ovarian cancer (OC). In this study, we found that miR-340 expression was downregulated in OC tissues compared with its expression in normal ovarian epithelium and endometrium, and treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) or trichostatin A (TSA) increased miR-340 expression in OC cells. In addition, ectopic miR-340 expression inhibited OC cell growth and metastasis in vitro and in vivo. Four and a half LIM domains protein 2 (FHL2) was confirmed as a direct target of miR-340 and silencing FHL2 mimicked the effects of miR-340 in OC cells. Further mechanistic study showed that miR-340 inhibited the Wnt/ß-catenin pathway by targeting FHL2, as well as downstream cell cycle and epithelial-to-mesenchymal transition (EMT) signals in OC cells. Moreover, the greatest association between miR-340 and FHL2 was found in 481 ovarian serous cystadenocarcinoma tissues via pan-cancer analysis. Finally, we revealed that lower miR-340 or higher FHL2 was associated with poor OC patient outcomes. Our findings indicate that the miR-340-FHL2 axis regulates Wnt/ß-catenin signaling and is involved in tumorigenesis in OC. Therefore, manipulating the expression of miR-340 or its target genes is a potential strategy in OC therapy.

The Lim1 oncogene as a new therapeutic target for metastatic human renal cell carcinoma.

Metastatic clear cell renal cell carcinoma (CCC) remains incurable despite advances in the development of anti-angiogenic targeted therapies and the emergence of immune checkpoint inhibitors. We have previously shown that the sonic hedgehog-Gli signaling pathway is oncogenic in CCC allowing us to identify the developmental Lim1 transcription factor as a Gli target and as a new oncogene in CCC regulating cell proliferation and apoptosis, and promoting tumor growth. In this previous study, preliminary in vitro results also suggested that Lim1 may be implicated in metastatic spread. Here we investigated the potential pro-metastatic role of Lim1 in advanced CCC (1) in vitro using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene either naturally or by gene transfer and (2) ex vivo in 30 CCC metastatic tissues, including lymph nodes, lung, skin, bone, and adrenal metastases, and (3) in vivo, using a metastatic model by intravenous injection of siRNA-transfected cells into Balb/c nude. Our in vitro results reveal that Lim1 knockdown time-dependently decreased CCC cell motility, migration, invasion, and clonogenicity by up to 50% regardless of their VHL status. Investigating the molecular machinery involved in these processes, we identified a large panel of Lim1 targets known to be involved in cell adhesion (paxillin and fibronectin), epithelial-mesenchymal transition (Twist1/2 and snail), invasion (MMP1/2/3/8/9), and metastatic progression (CXCR4, SDF-1, and ANG-1). Importantly, Lim1 was found constitutively expressed in all metastatic tissues. The H-score in metastatic tissues being significantly superior to the score in the corresponding primary tumor tissues (P value = 0.009). Furthermore, we showed that Lim1 silencing decreases pulmonary metastasis development in terms of number and size in the in vivo metastatic model of human CCC. Taken together, these experiments strengthen the potential therapeutic value of Lim1 targeting as a promising novel approach for treating metastatic human CCC.

microRNA-9 selectively targets LMX1A to promote gastric cancer cell progression.

LIM homeobox transcription factor 1, alpha (LMX1A) is downregulated in human gastric cancer (GC), functioning as a tumor suppressor. The current study aims to identify specific microRNA that can regulate LMX1A expression. By sequence analysis of LMX1A mRNA 3'-untranslated region (3'-UTR), we show that microRNA-9 (miR-9) putatively targets human LMX1A. In established (AGS cells) and primary human GC cells, ectopic overexpression of miR-9 by a lentiviral construct decreased LMX1A 3'-UTR activity, causing LMX1A mRNA and protein downregulation. Functional analyses show that miR-9 overexpression enhanced GC cell survival and proliferation. On the contrary, miR-9 inhibition by antagomir-9 lentivirus increased LMX1A 3'-UTR activity to upregulate LMX1A mRNA and protein expression, causing GC cell apoptosis. CRISPR/Cas9-mediated LMX1A knockout promoted AGS cell survival and proliferation. Importantly, miR-9 and antagomiR-9 were ineffective to the function of LMX1A-knockout AGS cells. In human GC tissues miR-9 is upregulated, which is negatively correlated with LMX1A downregulation. Together, we conclude that miR-9 selectively targets LMX1A to promote GC cell progression.

Increased expression of FHL2 promotes tumorigenesis in cervical cancer and is correlated with poor prognosis.

PURPOSE: Increasing evidence demonstrates that the four and a half LIM domain (FHL) gene and its protein products have different functions in the progression of various malignancies. However, the role of FHL protein 2 (FHL2) in cervical cancer (CC) has not been fully elucidated. In this study, we investigated the prognostic value of FHL2 expression in human CC tissues and the potential molecular mechanisms through which FHL2 modulates CC cell proliferation and apoptosis. MATERIALS AND METHODS: We measured FHL2 expression in CC cell lines and tissues by quantitative real-time polymerase chain reaction and Western blot assays. The effects of FHL2 knockdown on cell proliferation and apoptosis in two CC cell lines were examined using RNA interference, cell counting kit-8, Western blot and flow cytometry assays. Furthermore, we assessed phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) expression in two CC cell lines to determine whether the AKT/mTOR pathway is involved in the effects of FHL2 silencing on cell proliferation and apoptosis. Nude mice tumorigenicity experiments were also performed to evaluate the effects of FHL2 on HeLa cell growth in vivo. RESULTS: We found that FHL2 was significantly upregulated in CC cell lines and tissues. According to survival curves, high FHL2 expression levels in patients were correlated with poor prognosis. Moreover, by decreasing p-AKT and p-mTOR protein levels, silencing FHL2 significantly inhibited cell proliferation and induced apoptosis. FHL2 knockdown also induced apoptosis by increasing the Bax-to-Bcl2 ratio. By contrast, FHL2 overexpression significantly promoted cell proliferation. Finally, decreased tumour growth in an in vivo animal model also demonstrated the tumour-suppressing effects of FHL2 knockdown. CONCLUSION: Our findings indicate that FHL2 is an important prognostic factor in CC and that it plays a crucial oncoprotein role by promoting cell proliferation and inhibiting apoptosis in CC, possibly by targeting the AKT/mTOR pathway.

Zinc finger gene nolz1 regulates the formation of retinal progenitor cells and suppresses the Lim3/Lhx3 phenotype of retinal bipolar cells in chicken retina.

BACKGROUND: The zinc-finger transcription factor Nolz1 regulates spinal cord neuron development by interacting with the transcription factors Isl1, Lim1, and Lim3, which are also important for photoreceptors, horizontal and bipolar cells during retinal development. We, therefore, studied Nolz1 during retinal development. RESULTS: Nolz1 expression was seen in two waves during development: one early (peak at embryonic day 3-4.5) in retinal progenitors and one late (embryonic day 8) in newly differentiated cells in the inner nuclear layer. Overexpression and knockdown showed that Nolz1 decreases proliferation and stimulates cell cycle withdrawal in retinal progenitors with effects on the generation of retinal ganglion cells, photoreceptors, and horizontal cells without triggering apoptosis. Overexpression of Nolz1 gave more p27 positive cells. Sustained overexpression of Nolz1 in the retina gave fewer Lim3/Lhx3 bipolar cells. CONCLUSIONS: We conclude that Nolz1 has multiple functions during development and suggest a mechanism in which Nolz1 initially regulates the proliferation state of the retinal progenitor cells and then acts as a repressor that suppresses the Lim3/Lhx3 bipolar cell phenotype at the time of bipolar cell differentiation. Developmental Dynamics 247:630-641, 2018. © 2017 Wiley Periodicals, Inc.

Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro.

Zearalenone (ZEA) is an estrogenic mycotoxin mainly produced as a secondary metabolite by numerous species of Fusarium. Previous work showed that ZEA had a negative impact on domestic animals with regard to reproduction. The adverse effects and the mechanisms of ZEA on mammalian ovarian folliculogenesis remain largely unknown, particularly its effect on primordial follicle formation. Thus, we investigated the biological effects of ZEA exposure on murine ovarian germ cell cyst breakdown and primordial follicle assembly. Our results demonstrated that newborn mouse ovaries exposed to 10 or 30µM ZEA in vitro had significantly less germ cell numbers compared to the control group. Moreover, the presence of ZEA in vitro increased the numbers of TUNEL and γH2AX positive cells within mouse ovaries and the ratio of mRNA levels of the apoptotic genes Bax/Bcl-2. Furthermore, ZEA exposure reduced the mRNA of oocyte specific genes such as LIM homeobox 8 (Lhx8), newborn ovary homeobox (Nobox), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and factor in the germline alpha (Figlα) in a dose dependent manner. Exposure to ZEA led to remarkable changes in the Lhx8 3'-UTR DNA methylation dynamics in oocytes and severely impaired folliculogenesis in ovaries after transplantation under the kidney capsules of immunodeficient mice. In conclusion, ZEA exposure impairs mouse primordial follicle formation in vitro.

Evolved Repression Overcomes Enhancer Robustness.

Biological systems display extraordinary robustness. Robustness of transcriptional enhancers results mainly from clusters of binding sites for the same transcription factor, and it is not clear how robust enhancers can evolve loss of expression through point mutations. Here, we report the high-resolution functional dissection of a robust enhancer of the shavenbaby gene that has contributed to morphological evolution. We found that robustness is encoded by many binding sites for the transcriptional activator Arrowhead and that, during evolution, some of these activator sites were lost, weakening enhancer activity. Complete silencing of enhancer function, however, required evolution of a binding site for the spatially restricted potent repressor Abrupt. These findings illustrate that recruitment of repressor binding sites can overcome enhancer robustness and may minimize pleiotropic consequences of enhancer evolution. Recruitment of repression may be a general mode of evolution to break robust regulatory linkages.

The four and a half LIM domains 2 (FHL2) regulates ovarian granulosa cell tumor progression via controlling AKT1 transcription.

The four and a half LIM domains 2 (FHL2) has been shown to play important roles in the regulation of cell proliferation, survival, adhesion, motility and signal transduction in a cell type and tissue-dependent manner. However, the function of FHL2 in ovarian physiology and pathology is unclear. The aim of this study was to determine the role and functional mechanism of FHL2 in the progression of ovarian granulosa cell tumors (GCTs). Immunohistochemical analysis indicated that FHL2 was overexpressed in GCT tissues. Cellular localization of FHL2 in GCT cells was cell cycle dependent. Knockdown of FHL2 suppressed GCT cell growth, reduced cell viability and inhibited cell migration. Consistently, ectopic expression of FHL2 in GCT cells with very low endogenous FHL2 promoted cell growth, improved cell viability and enhance cell migration. Importantly, overexpression of FHL2 promoted GCT progression in vivo. Mechanistic studies indicated that FHL2 regulates AKT1 gene expression in vitro and in vivo. Knockdown of FHL2 or AKT1 in GCT cell lines induced very similar phenotypes. Ectopic expression of constitutively active AKT1 rescued FHL2 knockdown-induced arrest of GCT cell growth and reduction of GCT cell viability, suggesting that FHL2 regulates GCT cell growth and viability through controlling AKT1 expression. Finally, co-immunoprecipitation and chromatin immunoprecipitation analyses indicated that FHL2 functions as a co-activator of NFκB and AP-1 to regulate AKT1 gene transcription. In conclusion, results from the present study indicate that FHL2 exerts its oncogenic action in GCT cells via controlling AKT1 gene expression. FHL2 is a promising target for the development of novel drugs against ovarian granulosa cell tumor.

LHX6 inhibits breast cancer cell proliferation and invasion via repression of the Wnt/ß-catenin signaling pathway.

LIM homeobox domain 6 (LHX6), a member of the LHX family of proteins, has been implicated in cancer development. However, the involvement of LHX6 in the development of breast cancer remains unclear. In the present study, the epigenetic regulation, biological function and associated molecular mechanisms of LHX6 in breast cancer were analyzed. The expression levels of LHX6 were demonstrated to be markedly decreased in breast cancer tissues and cell lines. In addition, it was found that increased LHX6 expression in breast cancer cell lines inhibited cell proliferation and invasion. Furthermore, increased LHX6 expression significantly decreased the expression of ß­catenin in MDA­MB­231 breast cancer cells, and small interfering RNA­ß­catenin enhanced LHX6­induced inhibition of cell proliferation and invasion in MDA­MB­231 breast cancer cells. These results indicate that LHX6 inhibits breast cancer cell growth and invasion through suppression of the Wnt/ß­catenin signaling pathway. Thus, the present study provides a novel insight into the underlying mechanism of tumorigenesis in breast cancer, indicating the therapeutic potential of LHX6 in the treatment of breast cancer.

Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes.

PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

Inhibition of four-and-a-half LIM domain protein-2 increases survival, migratory capacity, and paracrine function of human early outgrowth cells through activation of the sphingosine kinase-1 pathway: implications for endothelial regeneration.

RATIONALE: Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair. OBJECTIVE: We investigated the role of FHL2 in the regulation of EOCs. METHODS AND RESULTS: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell-derived factor-1-induced EOC migration through upregulation of αv/ß3, αv/ß5, and ß2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2(-/-) mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2(-/-) versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2(-/-) versus WT EOCs. CONCLUSIONS: Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.

The LIM-only protein FHL2 attenuates lung inflammation during bleomycin-induced fibrosis.

Fibrogenesis is usually initiated when regenerative processes have failed and/or chronic inflammation occurs. It is characterised by the activation of tissue fibroblasts and dysregulated synthesis of extracellular matrix proteins. FHL2 (four-and-a-half LIM domain protein 2) is a scaffolding protein that interacts with numerous cellular proteins, regulating signalling cascades and gene transcription. It is involved in tissue remodelling and tumour progression. Recent data suggest that FHL2 might support fibrogenesis by maintaining the transcriptional expression of alpha smooth muscle actin and the excessive synthesis and assembly of matrix proteins in activated fibroblasts. Here, we present evidence that FHL2 does not promote bleomycin-induced lung fibrosis, but rather suppresses this process by attenuating lung inflammation. Loss of FHL2 results in increased expression of the pro-inflammatory matrix protein tenascin C and downregulation of the macrophage activating C-type lectin receptor DC-SIGN. Consequently, FHL2 knockout mice developed a severe and long-lasting lung pathology following bleomycin administration due to enhanced expression of tenascin C and impaired activation of inflammation-resolving macrophages.

A novel colon cancer gene therapy using rAAV­mediated expression of human shRNA-FHL2.

FHL2 (Four and a half LIM-only protein 2) has been identified as an oncogene in colon cancer and suppression of FHL2 induces cell differentiation and tumorigenesis in colon cancer cell lines. The aim of this study was to develop a novel and effective approach to knockdown FHL2, which can serve as a promising target of colon cancer therapy. Recombinant adeno-associated virus (rAAV) was generated bearing with FHL2-shRNA and transfected into LoVo cells. Cell cycle and growth were assessed. The interaction between FHL2 and G0/G1 cell cycle and growth was evaluated by flow cytometry, western blot analysis and WST-1 assay. We showed that suppression of FHL2 by rAAV-shRNA induced G0/G1 cell cycle arrest and inhibited cell growth. Apoptosis-related proteins and their activity was investigated at the same time. rAAV-FHL2­shRNA activated intrinsic and extrinsic apoptotic pathways and increased cell susceptibility to apoptotic stimuli by 5-FU. Moreover, a xenograft model was established to explore rAAV-FHL2-shRNA with 5-FU mediated tumorigenesis in vivo. A strong anti-tumorigenic effect of rAAV-FHL2-shRNA was shown in nude mice and this antitumor effect was enhanced when combined with 5-FU treatment. These findings implicate FHL2 as a cell cycle and growth modulator and thus inhibit apoptosis in colon cancer cells. rAAV-shRNA-FHL2 may serve as a novel and potent therapeutic or 5-FU co-therapeutic agent for colon cancer.

LHX2 regulates the neural differentiation of human embryonic stem cells via transcriptional modulation of PAX6 and CER1.

The LIM homeobox 2 transcription factor Lhx2 is known to control crucial aspects of neural development in various species. However, its function in human neural development is still elusive. Here, we demonstrate that LHX2 plays a critical role in human neural differentiation, using human embryonic stem cells (hESCs) as a model. In hESC-derived neural progenitors (hESC-NPs), LHX2 was found to be expressed before PAX6, and co-expressed with early neural markers. Conditional ectopic expression of LHX2 promoted neural differentiation, whereas disruption of LHX2 expression in hESCs significantly impaired neural differentiation. Furthermore, we have demonstrated that LHX2 regulates neural differentiation at two levels: first, it promotes expression of PAX6 by binding to its active enhancers, and second, it attenuates BMP and WNT signaling by promoting expression of the BMP and WNT antagonist Cerberus 1 gene (CER1), to inhibit non-neural differentiation. These findings indicate that LHX2 regulates the transcription of downstream intrinsic and extrinsic molecules that are essential for early neural differentiation in human.

FHL family members suppress vascular endothelial growth factor expression through blockade of dimerization of HIF1α and HIF1ß.

Four and a half LIM domain (FHL) proteins belong to a family of LIM-only proteins that have been implicated in the development and progression of various types of cancers. However, the role of FHL proteins in tumor angiogenesis remains to be elucidated. Herein, we demonstrate that FHL1-3 decrease the promoter activity and expression of vascular endothelial growth factor (VEGF), the key regulator of angiogenesis in cancer growth and progression as well as an important target gene of the transcription factor hypoxia-inducible factor 1 (HIF1α/HIF1ß). FHL1-3 interacted with HIF1α both in vitro and in vivo. A single LIM domain of FHL1 was sufficient for its interaction with HIF1α. FHL1 interacted with the HIF1α region containing basic helix-loop-helix (bHLH) motif and PER-ARNT-SIM domain, both of which aid in dimerization with HIF1ß and DNA binding. FHL1-3 inhibited HIF1 transcriptional activity and HIF1-mediated VEGF expression in a hypoxia-independent manner. Moreover, FHL1 blocked HIF1α-HIF1ß heterodimerization and HIF1α recruitment to the VEGF promoter. These data suggest that FHL proteins are involved in negative regulation of VEGF possibly by interfering with the dimerization and DNA binding of HIF1 subunits and may play an important role in tumor angiogenesis.

Chondrolectin mediates growth cone interactions of motor axons with an intermediate target.

The C-type lectin chondrolectin (chodl) represents one of the major gene products dysregulated in spinal muscular atrophy models in mice. However, to date, no function has been determined for the gene. We have identified chodl and other novel genes potentially involved in motor axon differentiation, by expression profiling of transgenically labeled motor neurons in embryonic zebrafish. To enrich the profile for genes involved in differentiation of peripheral motor axons, we inhibited the function of LIM-HDs (LIM homeodomain factors) by overexpression of a dominant-negative cofactor, thereby rendering labeled axons unable to grow out of the spinal cord. Importantly, labeled cells still exhibited axon growth and most cells retained markers of motor neuron identity. Functional tests of chodl, by overexpression and knockdown, confirm crucial functions of this gene for motor axon growth in vivo. Indeed, knockdown of chodl induces arrest or stalling of motor axon growth at the horizontal myoseptum, an intermediate target and navigational choice point, and reduced muscle innervation at later developmental stages. This phenotype is rescued by chodl overexpression, suggesting that correct expression levels of chodl are important for interactions of growth cones of motor axons with the horizontal myoseptum. Combined, these results identify upstream regulators and downstream functions of chodl during motor axon growth.

Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote epidermal regeneration after injury.

The Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives as well as in controlling stem cell activity. Here, we show that during murine skin morphogenesis, Lhx2 is expressed in the hair follicle (HF) buds, whereas in postnatal telogen HFs Lhx2(+) cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Remarkably, Lhx2(+) cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, as wound re-epithelization is significantly retarded in heterozygous Lhx2 knockout (+/-) mice, whereas anagen onset in the HFs located closely to the wound is accelerated compared with wild-type mice. Cell proliferation in the bulge and the number of Sox9(+) and Tcf4(+) cells in the HFs closely adjacent to the wound in Lhx2(+/-) mice are decreased in comparison with wild-type controls, whereas expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2(+/-) mice is inhibited by administration of Lgr5 siRNA. Finally, Chip-on-chip/ChIP-qPCR and reporter assay analyses identified Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as an important regulator of epithelial stem cell activity in the skin response to injury.